copyright © 2014, american society for microbiology. all...

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Assessment of influenza virus hemagglutinin stalk-based immunity in ferrets 1

Florian Krammer1, Rong Hai1, Mark Yondola1#, Gene S. Tan1, Victor Leyva-Grado1, Alex B Ryder2 , 2 Matthew S. Miller1, John K. Rose2, Peter Palese1,3, Adolfo García-Sastre1,3,4 and Randy A. Albrecht1,4* 3

1Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 4 2Department of Pathology, Yale University School of Medicine, New Haven, CT 5 3Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY 6 4Global Health & Emerging Pathogens Institute at Icahn School of Medicine, New York, NY 7 #Current affiliation: Avatar Biotechnologies, Brooklyn, NY 8 *To whom correspondence should be addressed: [email protected] 9 10

Abstract 11

Therapeutic monoclonal antibodies that target the conserved stalk domain of the influenza virus 12

hemagglutinin as well as stalk-based universal influenza virus vaccine strategies are being developed as 13

promising countermeasures for influenza virus infections. The pan-H1 reactive monoclonal antibody 14

6F12 has been extensively characterized and shows broad efficacy against divergent H1N1 strains in the 15

mouse model. Here we demonstrate its efficacy against a pandemic H1N1 challenge virus in the ferret 16

model of influenza disease. Furthermore, we recently developed a universal influenza virus vaccine 17

strategy based on chimeric hemagglutinin constructs that focuses the immune response towards the 18

conserved stalk domain of the hemagglutinin. Here we set out to test this vaccination strategy in the 19

ferret model. Both strategies, pre-treatment of animals with stalk-reactive monoclonal antibody as well 20

as vaccination with chimeric hemagglutinin based constructs were able to significantly reduce viral titers 21

in nasal turbinates, lungs and olfactory bulbs. In addition, vaccinated animals also showed reduced nasal 22

wash viral titers. In summary both strategies showed efficacy in reducing viral loads after influenza virus 23

challenge in the ferret model. 24

25

Importance 26

Influenza virus hemagglutinin-stalk reactive antibodies tend to be less potent, yet are more broadly 27

reactive and can neutralize seasonal and pandemic influenza virus strains. The ferret model was utilized 28

to assess the potential of hemagglutinin stalk-based immunity to provide protection against influenza 29

virus infection. The novelty and significance of the findings described in this manuscript support the 30

development of vaccines stimulating stalk-specific antibody responses. 31

32

Introduction 33

JVI Accepts, published online ahead of print on 8 January 2014J. Virol. doi:10.1128/JVI.03004-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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In the United States, epidemics of seasonal influenza cause substantial morbidity (46), and significant 34

mortality (7). Despite the proven ability of inactivated and live attenuated influenza virus vaccines to 35

reduce the impact of influenza, the potential of currently licensed influenza vaccines is not fully 36

manifested due to several factors. First, influenza vaccination coverage rates remain low (8). In 37

particular, a recent survey of 11,963 adults (18–64 years of age) revealed that only 28.2% reported 38

receiving the 2008–2009 influenza vaccine (72). Second, influenza vaccines induce immune responses 39

that specifically neutralize influenza viruses that are closely related to the vaccine strain, yet the potency 40

of these neutralizing responses diminishes with antigenic drift. Thus, annual influenza vaccination is 41

required to maintain protective immune responses against a “moving target” (66). Third, the 42

emergence of pandemic influenza virus strains is difficult to predict, and once an influenza pandemic 43

emerges it is even more difficult to redirect vaccine production in a timely fashion to respond to a 44

pandemic as happened during the 2009 H1N1 influenza pandemic (2, 9). Predictions of influenza 45

pandemics is further complicated by the realization that several influenza virus subtypes possess 46

pandemic potential, as evidenced by the emergence of avian influenza A (H7N9) virus in March 2013 47

(19), and sporadic human infections with H4, H5, H6, H7, H9 and H10 avian influenza viruses (1, 29, 31, 48

49, 63, 74). 49

50

Hemagglutinin (HA)-specific universal influenza vaccines have the potential to mitigate these limitations 51

by focusing humoral immune reponses on its antigenically conserved stalk region. Approaches to 52

developing stalk-focused universal vaccines have included headless HA (6, 51, 58), recombinant soluble 53

HA (20, 21, 32, 35, 41), synthetic polypeptides (67), prime boost regimens (69, 70) , nanoparticles (28), 54

and recombinant influenza viruses expressing chimeric hemagglutinin (21, 35). Stalk-specific vaccines 55

would shift the humoral immune responses away from the immunodominant globular head domain to 56

the more conserved stalk domain. Universal vaccines stimulating stalk-specific antibody responses 57

would have several desirable aspects, including: i) conferring protection against homologous and drifted 58

influenza virus strains, ii) obviating the need for annual influenza vaccinations with reformulated H1, H3, 59

and B virus strains that antigenically match prevalent circulating strains, and iii) conferring increased 60

protection against newly emerging influenza viruses with pandemic potential (33, 34). Importantly, stalk 61

reactive antibodies naturally occur in humans, albeit in general at low frequencies, and have been 62

detected in experimentally vaccinated mice (10, 14, 35, 36, 40, 44, 45, 50, 61, 73). Based on sequence 63

conservation, a universal influenza vaccine targeting the HA stalk would likely require three components 64


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to cover group 1 (H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17) and group 2 (H3, H4, H7, H10, H14, 65

H15) influenza A virus HAs, and influenza B virus HAs. 66

67

In this manuscript, we have examined in ferrets the level of protection conferred by group 1 HA stalk-68

specific antibodies against challenge infection with pandemic H1N1. Ferrets were passively immunized 69

with stalk-reactive monoclonal antibodies or were vaccinated with recombinant viral vectors expressing 70

chimeric HAs, known to induce stalk-reactive antibodies in mice. These studies revealed that group 1 71

stalk specific antibodies could reduce titers of infectious virus within the nasal cavity and also reduced 72

pulmonary virus titers in immunized ferrets challenged with a pandemic H1N1 influenza virus, which 73

contains an HA head not present in the chimeric HA vaccination regimen. These findings suggest that 74

ferrets produce HA stalk reactive antibodies following vaccination with chimeric HAs, and that stalk 75

reactive antibodies provide protection from high viral loads after challenge infection in this influenza 76

animal model. 77

78

Materials and Methods 79

Cells and viruses 80

Madin Darby canine kidney (MDCK), 293T, 293, A549 and BHK-21 cells were propagated in DMEM or 81

MEM medium (both Gibco). A/Netherlands/602/09 pandemic H1N1 virus and the recombinant B-cH9/1 82

virus (a B/Yamagata/16/88 virus that expresses a cH9/1 HA as described in (50)) were grown in 83

embryonated chicken eggs and titered on MDCK cells in media containing tosyl phenylalanyl 84

chloromethyl ketone (TPCK)-treated trypsin as described before. 85

Generation of a vesicular stomatitis virus (VSV) vector expressing cH5/1 protein 86

The cH5/1 gene (an A/Viet Nam/1203/04 H5 head on top of an A/PR/8/34 H1 stalk domain (21, 35)) was 87

amplified by PCR and the SalI-NheI restriction digested PCR product was then cloned into the XhoI and 88

NheI sites of the pVSV-XN2 (54) vector to generate pVSV-cH5/1. Recombinant vesicular stomatitis virus 89

expressing cH5/1 HA (VSV-cH5/1) was recovered using the above plasmid with minor modifications to 90

the previously described method (39). Briefly, baby hamster kidney-21 (BHK-21) cells were infected 91

with the T7 polymerase expressing vaccinia virus, vTF7-3 (18), at a multiplicity of infection (MOI) of 20. 92

At 1 h post-infection, the cells were transfected with the pVSV-cH5/1 plasmid and the support plasmids, 93

pBS-N, pBS-P, pBS-G and pBS-L. At 48 h post-transfection, the cell culture media was collected, filtered 94


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through a 0.1 μm filter and passaged onto BHK-21 cells. After cytopathic effect (CPE) became evident, 95

the culture media was collected, virus was plaque purified and used to grow up stocks. A VSV vector 96

expressing green fluorescent protein (GFP) was used as a control. 97

98

Generation of an adenovirus 5 vector expressing cH6/1 protein 99

Prior to virus generation, the cH6/1 (an A/mallard/Sweden/81/02 H6 globular head domain on top 100

of an H1/PR8 stalk domain (35, 50)) was cloned into a previously described transfer plasmid (pE1A-CMV, 101

lacking the HA epitope tag) (17). For virus generation, 2.0 X 106 human embryonic kidney (HEK) 293 cells 102

(generously supplied by Patrick Hearing) were plated per well of a 6-well dish and transfected the 103

following day with a 3:1 ratio of X-tremeGENE 9 (Roche):DNA according to the manufacturer’s 104

instructions. Cells were transfected with a total of 5.5 ug of DNA consisting of 5 ug of PvuI-linearized 105

cH6/1 pE1A-CMX plasmid and 500 ng of dl309 viral DNA that had been digested with ClaI/XbaI to 106

remove the left end of the adenoviral genome (bp 1-920). X-tremeGENE 9 -transfected, ClaI/XbaI-107

digested viral DNA was used as a negative control. After a 24 hour incubation, cells were overlayed with 108

2X DMEM-supplemented 1% agarose for plaque selection. Overlays were re-applied approximately 109

every 3 days for one week at which time plaques were isolated for screening and used for 10 lysate 110

generation. Once cytopathic effect (CPE) was evident (2-3 days), cells were harvested and frozen at -111

800C. Cells underwent 4 freeze/thaw cycles and then viral DNA was prepared by an established method 112

for sequencing (53). Once the cH6/1 sequence was confirmed, virus stocks were amplified on HEK-293 113

cells and purified by consecutive banding on step and equilibrium cesium chloride (CsCl) gradients. 114

Expression of the cH6/1 protein was confirmed by immunofluorescence staining on A549-infected cells 115

with anti-stalk mAb 6F12 (60) and virus titers were determined by standard plaque assay on HEK-293 116

cells. The empty control adenovirus vector (in the same genomic background) was kindly provided by 117

Patrick Hearing. 118

119

Immunostaining 120 121 MDCK cells were infected at a multiplicity of infection (MOI) of 1 with B-cH9/1 or wild type 122

B/Yamagata/16/88 and were fixed (0.5% para-formaldehyde) 24 hours post infection. A subset of cells 123

was permeabilized with 0.1% Triton X-100 and stained with an anti-influenza B nucleoprotein antibody 124

(Abcam, 1:1000). The rest of the cells were stained with anti-H1 stalk antibody 6F12 (10ug/ml) or anti-125


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H9 head antibody G1-26 (BEI Resources, NR-9485, 1:1000). 293T and A549 cells were 126

infected/transduced with empty or cH6/1 expressing adenovirus at an multiplicity of infection of about 127

100. Cells were permeabilized using 0.5% Triton X -100 and stained with an anti-hexon antibody (Abcam, 128

1:1000), anti-H1 stalk antibody 6F12 (10ug/ml) or anti-H6 head antibody NatalieC (10ug/ml). An 129

Alexa488 conjugated anti-mouse antibody (Life Technologies, 1:1000) was used as secondary antibody 130

for immunofluorescence. Vero cells were infected at a low multiplicity of infection with VSV viruses 131

expressing GFP or cH5/1 HA. Cells were fixed 24 hours post infection and stained with mouse anti-VSV 132

serum (1:1000), mAb 6F12 (10ug/ml) or anti H5 head antibody VN4-10 (BEI Resources, NR-2737, 133

1:1000). An anti-mouse horseradish peroxidase linked secondary (Santa Cruz, 1:3000) was used as 134

secondary antibody and stained cells were visualized using eminoethyl carbazole (AEC) substrate 135

solution (Millipore). 136

137

Antibodies and recombinant proteins 138

Mouse monoclonal antibodies 6F12 (H1 stalk-reactive, IgG2b) (60) and XY102 (A/Hong Kong/1/68 HA 139

head reactive, HI active, IgG2b)(47) were purified from supernatants of hybridoma cultures as described 140

before. Briefly, the supernatants were passed over a column loaded with protein G sepharose (GE 141

Healthcare), washed, eluted, concentrated and buffer exchanged to PBS (pH7.4) using Amicon Ultra 142

centrifugation units (Millipore). Protein concentration was determined with the A280 method using a 143

Nanodrop device. Recombinant HAs were expressed as ectodomains with a C-terminal trimerization 144

domain and hexahistidine tag using the baculovirus system as described before (32, 42). Protein 145

concentration was measured using the Bradford method. 146

Animals, passive transfer, immunization and challenge 147

Five month old male Fitch ferrets were confirmed seronegative for circulating H1N1, H3N2, and B 148

influenza viruses prior to purchase from Triple F Farms (Sayre PA). Ferrets were housed in PlasLabs 149

poultry incubators with free access to food and water (3, 43, 55). All animal experiments were 150

conducted using protocols approved by the Icahn School of Medicine at Mount Sinai Institutional Animal 151

Care and Use Committee (IACUC). Animals were anesthetized by intramuscular administration of 152

ketamine/xylazine for all described procedures including bleeding, nasal washes, vaccination, infection 153

and passive transfer. 154


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For passive transfer experiments animals were bled to obtain baseline sera two weeks before the 155

transfer. On day -1, 30 mg/kg of mouse mAb 6F12 or XY102 (n=2 per group) were transferred 156

intravenously via the vena cava (Figure 1A). At 24 hours post inoculation, animals were bled and 157

infected with 104 plaque forming units (PFU) of A/Netherlands/602/09 (pandemic H1N1) virus. Nasal 158

washes were then taken on day 1 and 3 post infection and body weights monitored daily. Animals were 159

observed for approximately 30 minutes daily for signs of morbidity (e.g. sneezing). On day 4 post 160

infection animals were sacrificed, exsanguinated and tissue samples from the upper left and right lobes 161

of the lung, olfactory bulb and nasal turbinates were taken. 162

For vaccination experiments animals (n=5) were intranasally infected with 2x107 PFU (in 1 ml of PBS) of 163

an influenza B virus vector B-cH9/1 HA (an H9 head on top of an H1 stalk domain (35, 50)) (Figure 4A). 164

Three weeks post infection animals were boosted by intramuscular administration of 2x105 PFU (in 0.5 165

ml) of a recombinant VSV- cH5/1 HA (an H5 globular head domain on top of an H1 stalk domain (21, 166

35)). A second boost consisting of replication deficient recombinant adenovirus 5 vector expressing 167

cH6/1 protein (an H6 globular head domain on top of an H1 stalk domain) was given intranasally and 168

intramuscularly (1.2x108 PFU in 0.5 ml per site) three weeks after the first boost. Control group animals 169

received the same empty or GFP expressing (VSV) virus vectors in the same sequence (n=4). Four weeks 170

after the last prime animals were challenged with 104 PFU of A/Netherlands/602/09 (pandemic H1N1) 171

virus. Nasal washes were then taken on day 1 and 3 post infection and weight was monitored daily. 172

Animals were observed for approximately 30 minutes daily and signs of morbidity (e.g. sneezing) were 173

recorded. On day 4 post infection animals were sacrificed and tissue samples from the lung (upper right 174

lobe), olfactory bulb and nasal turbinates were taken. 175

Hemagglutination inhibition assay 176

Hemagglutination inhibition (HI) assays were performed as described elsewhere (3, 8). Working 177

stocks for each influenza virus strain were prepared by diluting the virus stock to a final HA titer of 8 HA 178

units/50μL. Each serum sample was serially diluted two-fold in PBS (25 μL per well) in 96 V-well 179

microtiter plates. Then, 25 μL of working stock of influenza virus strain was added to each well so that 180

all wells contain a final volume of 50 μL. The serum-virus samples were then incubated at room 181

temperature for 45 minutes to allow HA head-specific antibodies to neutralize the influenza virus. To 182

each well, 50 μL of a 0.5% suspension of turkey or chicken red blood cells was added. The assay plates 183

were then incubated at 4°C until red blood cells in the PBS control sample formed a button, and red 184


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blood cells hemagglutinated in control wells containing virus and no antibody. The HI titer was defined 185

as the reciprocal of the highest dilution of antibody that inhibits red blood cell hemagglutination by 186

influenza virus. 187

188

Enzyme linked immunosorbent assay 189

Enzyme linked immunosorbent assays (ELISA) were performed as described before (32, 36). Briefly, 190

plates were coated with 2 ug/ml of recombinant, baculovirus produced H1 (from A/California/04/09, 191

A/New Caledonia/20/99 and A/South Carolina/1/18), H2 (from A/Japan/305/57) or H17 (from A/yellow 192

shouldered bat/Guatemala/06/10) HA proteins (32). Wells were then incubated with serial (2-fold) 193

dilutions of ferret sera, nasal washes or lung homogenates for 1 hour at room temperature. After 194

extensive washes plates were incubated with anti-mouse (for mAbs, Santa Crus) or anti-ferret (Alpha 195

Diagnostics Intl.) IgG horse radish peroxidase (HRP) labeled secondary antibodies for another hour at RT. 196

Plates were washed again and were then developed using SigmaFast ODP substrate and read on a 197

Synergy H1 (BioTek) plate reader. 198

199

Results 200

Persistence and tissue distribution of 6F12 in the ferret 201

Previously, we have shown that HA head-reactive IgA but not IgG antibody is able to prevent 202

transmission in the ferret and guinea pig model of influenza infection (56). We reasoned that at 203

especially low concentrations (3 mg/kg), IgG is not efficiently transported to mucosal surfaces. This 204

transport might be additionally inhibited by the lower Fc-Fc-receptor interactions between mouse mAbs 205

and the ferret host. In addition, the half-life of mouse IgG in ferrets has not been well characterized; 206

however, a previous study that examined the therapeutic potential of a humanized mAb, m102, in the 207

ferret model of Nipah virus infection reported an elimination half-life of 3.5 days following intravenous 208

administration of 25 mg of mAb (75). We were therefore curious if treatment with a high dose of mAb 209

(30 mg/kg) would increase the Ab concentration in mucosal surfaces and protect from upper respiratory 210

tract infection. Ferrets were passively immunized by intravenous administration of 30 mg/kg of either 211

H1 stalk-specific mAb 6F12 or H3-specific mAb XY102 (isotype control) (Figure 1A). The persistence and 212


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tissue distribution of mAb 6F12 was examined by ELISA with baculovirus-produced H1 from 213

A/California/04/09. mAb 6F12 could be easily detected by ELISA within serum samples at day 4 post-214

passive immunization (Figure 1B). In addition, mAb 6F12 was detected by ELISA in nasal wash samples 215

collected at day 1 but the level of mAb declined by day 3 post-challenge infection (Figure 1C). mAb 6F12 216

was also detected by ELISA in lung homogenates at day 4 post-challenge (Figure 1D). These results 217

suggest that passive immunization by intravenous administration of mAb 6F12 would confer a window 218

of protection within the respiratory tract of ferrets against challenge infection. 219

Prophylactic administration of 6F12 reduces viral loads in lungs, olfactory bulbs and nasal turbinates 220

Mouse monoclonal antibody 6F12 is an H1 stalk domain specific antibody that potently inhibits viral 221

replication of H1N1 virus isolates spanning from 1930 to 2009 and efficiently protects mice 222

prophylactically and therapeutically from viral challenge (60). In order to investigate whether 6F12 223

would also be efficacious prophylactically in the ferret model of influenza disease, this mAb was 224

administered to ferrets intravenously at a 30 mg/kg dose 24 hours pre-challenge and animals were then 225

challenged with the pandemic H1N1 strain A/Netherlands/602/09. Control group ferrets received the 226

same amount of an isotype control antibody (Figure 1A). Viral titers from nasal washes taken on day 1 227

and day 3 were slightly lower in the 6F12-treated animals as compared to the control group (Figure 2A). 228

The effect was more pronounced on day 1 than on day 3, which matches with the lower 6F12 titers 229

found in nasal washes on day 3 post-challenge. Furthermore, day 4 nasal turbinate titers of 6F12-treated 230

ferrets were lower than titers of control animals (Figure 2B). A reduction of approximately two logs for 231

6F12-treated animals was also observed in the olfactory bulb (Figure 2C) and lung titers were 232

approximately one log lower as compared to control animals (Figure 2D). Weight loss was only minimal 233

and similar in both groups (data not shown). In summary, prophylactic treatment of ferrets with mAb 234

6F12 reduced the viral load in challenged animals in all analyzed tissues. The readouts established for 235

this experiment were then also used to compare and analyze efficacy of a chimeric HA (cHA) vaccine 236

regimen in ferrets. 237

Vaccination with chimeric hemagglutinins induces stalk-reactive antibodies in the ferret 238

We have previously shown that vaccination of inbred BALB/c mice with chimeric HA constructs (HAs 239

with a conserved stalk domain but divergent head domains) induce broadly neutralizing stalk-reactive 240

antibodies (35). Here we wanted to test if vaccination of ferrets would also induce stalk-reactive 241


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antibodies. To this end, we utilized viral vectors expressing chimeric HA constructs (Figure 3). Prior to 242

vaccination of ferrets with the viral vectors, the expression of the chimeric HA was demonstrated by 243

immunostaining. The expression of chimeric hemagglutinin by an influenza B virus vector B-cH9/1 HA 244

(35, 50) was demonstrated by immunofluorescence assay with infected MDCK cells (Figure 3A). The 245

expression of chimeric hemagglutinin by a VSV-cH5/1 HA (21, 35) was demonstrated by immunostaining 246

of virus plaques in Vero cells (Figure 3B). The expression of chimeric hemagglutinin by a replication 247

deficient adenovirus 5 vector expressing cH6/1 HA (an H6 head on top of an H1/PR8 stalk domain (21, 248

35)) was demonstrated by immunofluorescence assay with infected 293T and A549 cells (Figure 3C). 249

250

Ferrets were first vaccinated with B-cH9/1 HA, and were then boosted with a VSV-cH5/1 HA (an H5 head 251

on top of an H1 stalk domain (21, 35)) and then with a replication deficient adenovirus 5 vector 252

expressing cH6/1 HA (an H6 head on top of an H1/PR8 stalk domain (21, 35)) (Figure 4A). This 253

vaccination regimen was chosen in order to avoid the generation of antibodies against any antigen in 254

pandemic H1N1 virus different from the HA stalk, which could also contribute to protection after 255

subsequent challenge. Vaccinated animals developed low sero-reactivity against pandemic H1 HA after 256

the prime. This reactivity was boosted approximately four-fold by the cH5/1 vaccination and then again 257

eight-fold by the final cH6/1 vaccination. Sera from vector control animals exhibited only background 258

reactivity that was comparable to the reactivity of pooled pre-vaccination sera of the used ferrets. Since 259

cHA vaccinated animals were naive to the H1 head domain and also tested HI negative against the 260

pandemic H1N1 strain A/Netherlands/602/09 we conclude that any reactivity to H1 strains is based on 261

cross-reactive antibodies to the conserved stalk domain. Furthermore our cHA vaccine constructs are 262

based on the stalk domain of A/PR/8/34 H1 HA. Therefore reactivity to pandemic H1 HA already 263

represents heterologous stalk reactivity within H1 HAs. We also tested reactivity to two more H1 HA, the 264

HA from the pre-pandemic seasonal strain A/New Caledonia/20/99 and the HA from the 1918 pandemic 265

H1N1 strain A/South Carolina/1/18 (Figure 4C-D). Sera from cHA-vaccinated animals reacted strongly 266

with both proteins. In order to test if cHA vaccination induces crossreactivity to other group 1 subtypes, 267

we also tested reactivity against an H2 HA from A/Japan/305/57 virus (Figure 4E) and against an H17 HA 268

(from the recently discovered bat H17N10 influenza virus strain A/yellow shouldered 269

bat/Guatemala/06/10) (Figure 4F). Sera from cHA vaccinated ferrets reacted strongly with both HAs 270

while sera from vector control animals showed only background reactivity (Figure 4E-F). Cross reactivity 271


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against group 2 HA was not expected since earlier studies in mice have shown that group 1 stalk based 272

cHA vaccination regimens do not protect from group 2 virus challenge and vice versa (35, 41). 273

Importantly, we did not detect any H1 head specific antibody responses against the challenge virus 274

following the vaccination regimen as measured by HI assay (data not shown). As positive controls, 275

convalescent reference sera from two ferrets that were infected with A/California/7/2009 were included 276

in the HI assay, and each reference serum yielded HI titers of 1280. 277

Chimeric hemagglutinin vaccine constructs protect ferrets from viral challenge. 278

In order to test the protection that cHA vaccination would confer to ferrets we challenged the animals 279

with the pandemic H1N1 strain A/Netherlands/602/09 (Figure 5A). The readouts were the same as for 280

the passive transfer experiment; we measured virus titers in day 1 and day 3 nasal washes as well as in 281

lung, olfactory bulb and nasal turbinates on day 4 post infection. Interestingly, nasal wash titers were 282

lower in cHA-vaccinated ferrets than in control animals on day 1 (approximately 5-fold) as well on day 3 283

(more than 10-fold) when the difference was highly significant (p=0.0005) (Figure 5A). This result is not 284

surprising since we expected that the intranasally applied prime and second boost would induce stalk-285

reactive mucosal IgA antibodies. The reduction of virus titers in the nasal washes is also reflected by a 286

significant reduction of virus titers in the nasal turbinates of about 10 fold (p=0.0331) (Figure 5B). 287

Furthermore, olfactory bulb titers of cHA-vaccinated animals were more than 2 logs lower as compared 288

to vector control animals (p=0.0062) (Figure 5C). In fact we were unable to detect virus in the olfactory 289

bulb of 4 out of 5 cHA-vaccinated ferrets whereas high virus titers were found in olfactory bulbs of all 4 290

control ferrets. Finally, we also detected a reduction of approximately half a log of lung virus titers in 291

cHA-vaccinated ferrets as compared to vector control ferrets (Figure 5D). In summary, the protective 292

efficacy of the cHA vaccine was comparable (nasal turbinates, olfactory bulbs and lung titers) or better 293

(nasal wash titers) as compared to the protective efficacy of prophylactically administered mAb 6F12. 294

Discussion 295

In recent years broadly neutralizing antibodies against the conserved stalk domain of the influenza virus 296

HA have been isolated (12-15, 22, 40, 59-62, 68, 73). These antibodies can be used for prophylactic as 297

well as therapeutic treatment of influenza virus infections. Although the high amount of mAb needed for 298

treatment might preclude the use of the antibodies in the general population, this approach might be 299

useful for the therapy of severe influenza cases especially when drug resistant viruses in an immune-300


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compromised host are involved (11, 25-27, 57, 64, 65). We therefore wanted to evaluate mAb 6F12 in a 301

prophylactic setting in the ferret model. This antibody has pan-H1 neutralizing activity in vitro and is able 302

to protect mice from challenge with H1N1 influenza viruses that span almost hundred years of antigenic 303

drift (60). We show here that mAb 6F12 is indeed efficacious against a pandemic H1N1 strain in the 304

ferret model as well. In particular, prophylactic administration of mAb 6F12 resulted in more 305

pronounced reduction in virus titers in olfactory bulbs and lungs. Unexpectedly, we could also detect 306

this mouse IgG antibody at low titers in nasal washes of treated ferrets. These low levels of antibody 307

found in the nasal washes correlated well with small reductions of nasal wash viral titers. Several factors 308

could contribute to the pronounced reduction of virus titers in olfactory bulb and lung samples 309

compared to the modest reduction of virus titers observed in nasal wash samples. At day 4 post-310

intravenous injection, high levels of mAb 6F12 could be detected in serum and lung samples which 311

contrasts with the low level of mAb 6F12 detected in nasal washes. In addition, mAb 6F12 liberated by 312

homogenizing of olfactory bulb and lung tissue samples would bind to and neutralize a small fraction of 313

virus present in the tissue samples prior to titration of virus titers by plaque assays. We speculate that 314

6F12-like antibodies, if transported efficiently to mucosal surfaces (e.g. locally induced by intranasally 315

administered vaccines) would be able to efficiently reduce nasal wash titers and possibly have an impact 316

on transmission as well. We recently showed that this is the case for globular head-reactive mAb 30D1 317

which efficiently blocks replication when administered to guinea pigs as IgA (efficiently transported to 318

mucosal surfaces) but lacks efficacy when administered as IgG (not efficiently transported to mucosal 319

surfaces) (56). 320

In an 'antibody-guided' vaccine approach based on stalk-reactive antibodies we have developed 321

chimeric HA vaccine constructs (21, 35). These constructs possess a conserved, structurally integrated, 322

stalk domain in combination with divergent globular head domains from 'exotic' subtypes (35). By 323

sequentially immunizing with these constructs, we protected mice from challenge with heterologous 324

(H1N1) and heterosubtypic (other group 1 HA expressing viruses) influenza viruses (35). Here, we 325

wanted to test the efficacy of this vaccine approach in the ferret model. By immunizing ferrets with 326

combinations of divergent globular heads and a conserved stalk domain, we hoped to get an immune 327

response that is focused on broadly neutralizing epitopes in the stalk. This strategy is based on the 328

observation that sequential infection/vaccination with seasonal H1N1 and pandemic H1N1 viruses 329

(which have highly divergent globular head domains and highly conserved stalk domains) induces high 330

levels of stalk-reactive antibodies in humans (40, 50, 52, 61, 73). Similar findings were also made in the 331


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mouse model (36). Here in the ferret model, we show that a cHA based immunization strategy confers 332

protection against pandemic H1N1 challenge. The observed level of protection was similar to or better 333

than protection conferred by inactivated, antigenically matched, unadjuvanted split vaccine 334

administered once (16, 24) or twice (4) or an antigenically matched experimental vaccinia virus 335

vectored construct (38). It is of note that the cHA-based vaccine did not induce any HI active antibodies, 336

but vaccinated ferrets were able to produce a broadly reactive anti-stalk response against divergent 337

group 1 HA subtypes. This proof of principle study focuses on protection afforded by the stalk domain of 338

HA. A human vaccine candidate based on the same principle would most likely consist of inactivated or 339

attenuated cHA expressing viruses that also have a neuraminidase (NA). We would argue that the 340

antibody titers against the more conserved NA would be boosted as well in the absence of an 341

immunodominant globular head domain (30, 48). These antibodies would then also contribute to broad 342

protection. Furthermore, conserved internal proteins like the nucleoprotein induce strong protective T-343

cell responses that contribute to protection as well (5, 23, 37, 71). We have conclusively shown that 344

such a vaccination strategy based on the H1 HA stalk domain is able to broadly protect against group 1 345

HA expressing viruses in mice but was unable to protect against an H3N2 challenge virus (35). We 346

therefore believe that a successful vaccination strategy in humans would need to contain a group 1, a 347

group2 and an influenza B virus stalk component to induce broadly neutralizing stalk antibodies. 348

In summary we have shown that treatment of ferrets with stalk reactive antibody as well as vaccination 349

with a stalk-based vaccination strategy is efficacious against influenza virus challenge in ferrets. We 350

believe that both strategies are valuable additions to the armamentarium for fighting seasonal and 351

pandemic influenza virus infections in the human population. 352

353

Acknowledgements 354

We thank Chen Wang and Richard Cadagan for excellent technical assistance. This study was partially 355

funded by a National Institutes of Health/National Institute of Allergy and Infectious Disease program 356

project grant (P01AI097092), by PATH, and by R01-AI080781. Florian Krammer was supported by an 357

Erwin Schrödinger fellowship (J 3232) from the Austrian Science Fund (FWF). Matthew S. Miller was 358

supported by a Canadian Institutes of Health Research Postdoctoral Fellowship. 359

360


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361


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Figure 1. Persistence and distribution of mAb 6F12 in passively immunized ferrets (n = 2 ferrets). (A) 362

Schematic representation of the passive immunization and challenge study. A baseline serum sample 363

was collected prior to passive immunization of ferrets with 30mg/kg of mAb 6F12 at day -1. At day 0 364

post-immunization, a serum sample was taken and ferrets were challenged by infection with 104 PFU of 365

A/Netherlands/602/09. (B) Titers of mAb 6F12 in serum samples collected at days -1, 0, and 4 of passive 366

immunization were measured by ELISA reactivity against baculovirus-produced H1 from 367

A/California/04/09. (C) Titers of mAb 6F12 in nasal wash samples collected at days 1 and 3 of passive 368


A/California/04/09. (D) Titers of mAb 6F12 in lung homogenate samples collected at day 4 of passive 370


A/California/04/09. For (C) and (D), nasal wash or lung samples from ferrets passively immunized with 372

mAb XY102 that specifically recognizes the H3 of A/Hong Kong/1/1968 served as negative controls (n = 2 373

ferrets). 374

375

Figure 2. Prophylactic administration of mAb 6F12 reduced viral titers following challenge infection. 376

Ferrets were passively immunized with mAb 6F12 (green bars; n = 2 ferrets) or isotype control mAb 377

XY102 (black bars; n = 2 ferrets). At day 0 post-passive immunization, ferrets were challenge infected 378

with 104 PFU of A/Netherlands/602/09. (A) Titers of challenge virus in nasal wash samples collected at 379

day 1 or 3 post-challenge infection were determined by plaque assay. At day 4 post challenge infection, 380

titers of influenza virus in nasal turbinates (B), olfactory bulb (C), and lung samples (D) were assessed by 381

plaque assays. 382

383

Figure 3. Expression of cHAs by viral vectors. (A) An engineered influenza B virus expresses cH9/1 HA (H9 384

head on top of an H1 stalk domain) instead of an influenza B HA. The panel shows staining of B-cH9/1 or 385

B wt infected cells with an anti-influenza B nucleoprotein antibody (anti-NP), anti H1 stalk antibody 6F12 386

(anti-stalk) or an anti H9 head antibody (anti-H9 head). (B) A recombinant VSV virus was engineered to 387

express cH5/1 (H5 head on top of an H1 stalk domain) HA as transgene. The panel shows staining of 388

VSV-cH5/1 or VSV-GFP infected Vero cells with anti-VSV mouse serum (anti-VSV), anti H1 stalk antibody 389

6F12 (anti-stalk) or an anti H5 head antibody (anti-H5 head). (C) A replication deficient adenovirus was 390

engineered to express cH6/1 HA (H6 head on top of an H1 stalk domain). The panels shows infected 391


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293T cells stained for the presence of adenovirus (anti-hexon) and transduced A459 cells stained with 392

anti H1 stalk antibody 6F12 (anti-stalk) or an anti H6 head antibody (anti-H6 head). 393

394

395

Figure 4. Ferrets develop HA stalk-specific humoral responses by repeated immunization with viral 396

vectors expressing chimeric hemagglutinins. (A) Schematic representation of the HA stalk-based 397

vaccination strategy. Ferrets (n = 5) were vaccinated with expressing-cH9/1 HA, boosted with VSV- 398

cH5/1 HA, and boosted a second time with adenovirus 5 vector expressing cH6/1 protein. Control 399

ferrets (n = 4) were vaccinated with wild-type influenza B virus or VSV (expressing GFP) and adenovirus 400

(empty) vectors. Ferrets were then challenged by infection with 104 PFU of A/Netherlands/602/09 401

virus. The development of broadly cross-reactive stalk specific antibody responses were assessed by 402

ELISA with baculovirus-produced H1 from A/California/04/09 (B), H1 from A/South Carolina/1/18) (C), 403

H1 from A/New Caledonia/20/99 (D), H2 ( from A/Japan/305/57) (E), or H17 (from A/yellow shouldered 404

bat/Guatemala/06/10) (F). 405

406

Figure 5. The HA stalk-based vaccination strategy confers protection against challenge infection with 407

A/Netherlands/602/09 virus. Ferrets (n = 5) were vaccinated with B-cH9/1 HA, boosted with VSV-cH5/1 408

HA, and boosted a second time with adenovirus 5 vector expressing cH6/1 protein. Control ferrets (n = 409

4) were vaccinated with wild-type influenza B virus or VSV (expressing GFP) and adenovirus (empty) 410

vectors. (A) Titers of challenge virus in nasal wash samples collected at day 1 or 3 post-challenge 411

infection were determined by plaque assay. At day 4 post challenge infection, titers of influenza virus in 412

nasal turbinates (B), olfactory bulb (C), and lung samples (D) were assessed by plaque assays. 413

414

415

416


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